Project Summary/Abstract Increased paracellular permeability is a hallmark of acute lung injury and the mechanisms underlying failure of both epithelial and endothelial barriers need to be further investigated. High morbidity and mortality in patients with acute lung injury is characterized by flooding of the airspaces with protein-rich edema. We previously reported that Rho GTPase activation plays a critical role in permeability derangements and actin stress fiber formation in lung microvascular endothelial and alveolar epithelial cells. Additionally, inhibition of Rho GTPase preserved the integrity of lung endothelial and alveolar epithelial barrier function and promoted alveolar fluid clearance in a murine model of Pseudomonas aeruginosa (P. aeruginosa) pneumonia. However, the molecular downstream mechanism(s) by which Rho GTPases alter cytoskeletal dynamics of lung microvascular endothelial and alveolar epithelial cells, and causes paracellular permeability derangements, is not fully understood. Active small Rho GTPase unlocks Neuronal Wiskott?Aldrich syndrome protein (NWASP) from an autoinhibited conformation for activation. NWASP transmits upstream signals to the cellular machinery directly involved in modulation of cytoskeletal dynamics. Our preliminary data and published work indicate that (a) NWASP downregulation inhibits actin stress fiber formation and reduces paracellular permeability in lung microvascular endothelial and alveolar epithelial cells after exposure to P. aeruginosa or TGF-?1; (b) Furthermore, inhibition of N-WASP protects mice against lung injury and improves survival in a murine model of P. aeruginosa pneumonia. We hypothesize that activation of the GBD domain of NWASP functions as a critical switch (the ?GBD-switch?) in the development of lung edema associated with bacteria-induced lung injury, through recruiting downstream signaling molecules, promoting cytoskeletal dynamics, and disrupting barrier function of both lung endothelium and alveolar epithelium. To test this hypothesis, we propose three specific aims: (Aim 1) to define the downstream partner(s), and critical domain(s), of NWASP that are essential to promote lung microvascular endothelial and alveolar epithelial permeability induced by P. aeruginosa; (Aim 2) to determine the common, and differential, molecular mechanisms, by which the activated GBD switch alters cytoskeletal dynamics, disrupts barrier function, and causes paracellular permeability in lung microvascular endothelial cells and alveolar epithelial cells in response to P. aeruginosa; (Aim 3) to define the in vivo, and cell-type-specific, role of NWASP in a murine model of acute lung injury caused by P. aeruginosa pneumonia. The findings from these studies will help to delineate the molecular mechanisms regulating cytoskeletal dynamics, barrier function, and permeability in P. aeruginosa-induced acute lung injury.